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Serological diagnosis of fascioliasis
Veterinary Parasitology, 1(1976) 239--248 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
SEROLOGICAL DIAGNOSIS OF FASCIOLIASIS
L.J. VAN TIGGELE and H.J. OVER Central Veterinary Institute, Lelystad (The Netherlands) (Received July 14th, 1975)
ABSTRACT Van Tiggele, L.J. and Over, H.J., 1976. Serological diagnosis of fascioliasis. Vet. Parasitol., 1: 239--248. Antibodies against Fasciola hepatica were detected in sera from experimentally infected sheep and calves. Titres in sera from field-infected sheep were related to fluke burdens. Indirect haemagglutination, counter-immunoelectrophoresis and double immunodiffusion were compared. Indirect haemagglutination and counter-immunoelectrophoresis were superior to double immunodiffusion and positive reactions in these techniques preceded the presence of parasite eggs in the faeces by at least 50 days. The relative advantages of the immunological techniques are discussed.
INTRODUCTION During t h e p a s t d e c a d e s c a t t e r e d i n f o r m a t i o n has b e c o m e available o n m e t h o d s o t h e r t h a n c o p r o l o g i c a l analysis f o r t h e diagnosis o f i n f e c t i o n w i t h Fasciola hepatica ( G e y e r , 1 9 6 7 ; G u n d l a c h , 1971). During t h e p r e p a t e n t period, a n d at l o w levels o f a d u l t f l u k e b u r d e n in p a r t i c u l a r , t h e n e e d f o r such t e c h n i q u e s is clear {H~rchner, 1 9 7 3 ; H e n r i k s e n , 1974). In e x p e r i m e n t a l i n f e c t i o n s as well as in field cases d o u b l e i m m u n o d i f f u s i o n {DID) p r o v e d t o be reliable, a l t h o u g h s o m e w h a t insensitive ( T r a w i n s k y , 1 9 3 7 ; K o c h , 1 9 6 9 ; Doyle, 1973). T o o b t a i n higher sensitivity we a d o p t e d c o u n t e r - i m m u n o e l e c t r o p h o r e s i s (CIE) a n d i n d i r e c t h a e m a g g l u t i n a t i o n ( I H A ) . C I E is an i m p r o v e m e n t on D I D a n d has also b e e n a p p l i e d t o c e r t a i n viral diseases s u c h as s e r u m h e p a t i t i s in m a n a n d A l e u t i a n disease in m i n k ( G o c k e and Howe, 1970; Cho and Ingram, 1972). I H A is a useful m e t h o d f o r large scale serological analysis in m i c r o t i t r e s y s t e m s . I t has b e e n a p p l i e d in a wide r a n g e o f parasitic i n f e c t i o n s in m a n (Kagan, 1 9 7 4 ) . Since serological m e t h o d s c a n b e s i m p l y u s e d t o t e s t a large n u m b e r o f sera, a t t e m p t s w e r e m a d e t o i m p r o v e a n d e v a l u a t e t h e s e useful t e c h n i q u e s .
240
MATERIAL AND METHODS
Hosts Blood samples were collected at necropsy from 122 Texel sheep, 7--14 months of age. They had grazed previously in groups of four for 2--3 months on fields with different infection levels and were kept for 3 months on fluke-free fields after this period. Thus at necropsy the infections were 3--6 months old. Flukes were collected from these sheep using previously described techniques (Dijkstra et al., 1973). To analyse the development of antibody titres during the course of infection, four 1-year-old sheep raised parasite free were infected with 500 metacercariae each. Infection was monitored by regular faecal examination and standard haematological procedures. During the period following infection, serum samples were analysed weekly for 8 weeks, then every 3 weeks for 18 weeks. After 26 weeks the sheep were killed and flukes were recovered and counted. In an experiment on calves, four 8-month-old animals were infected with 2 000 metacercariae each. The animals were kept in normal farm conditions on fluke-free fields. During the course of the experiment, observations were made every 10 or 11 days with regard to egg production, serology and haematology. After 15 weeks serological examination was performed every 21 days. The animals were necropsied after 26 weeks and flukes were collected. All sera were stored at --20°C until examination.
Infective material In the artificial infection experiment, a fluke strain obtained by cloning was used. Cercariae were obtained from Lymnaea truncatula at 21°C. They were collected according to Boray's (1963) method, but instead of encysting on glass the metacercariae were collected on cellophane paper. Metacercariae were stored at 4°C {relative h u m i d i t y 100%) and were 3--8 weeks of age at the time of infection. Animals were infected orally by administration of gelatine capsules containing the metacercariae.
Faecal examination Faeces samples were examined according to the m e t h o d described by Dorsman {1956).
Antigens Flukes were obtained from sheep livers at a slaughterhouse and a standard lipid-free antigen was prepared according to Kent (1960).
241 Protein c o n t e n t was estimated using the biuret m e t h o d as described by Kabat (1961). Antigen containing 4.5 mg protein/ml was stored a t - - 2 0 ° C t h r o u g h o u t the experiment.
Indirect haemagglutination (IHA ) Sheep erythrocytes were fixed as described by Herbert (1967) using glutaraldehyde instead of formaldehyde. A 1% suspension of these stabilized cells was sensitized with tannic acid 1 : 40 000. To prevent clumping, tanned cells were pre-coated by mixing equal volumes of 2% tanned cells in 0.1 M phosphate buffered saline (PBS) pH 7.2 with 0.5% bovine serum albumin in PBS (Eidinger, 1964). Antigens were attached at pH 6.4 using 0.2 ml of standard antigen (4.5 mg/ml) per 10 ml of 1% cells (30 min, 37°C). Antigen-coated cells were stored at 4°C in 1% inactivated normal rabbit serum in PBS with 0.02% sodium azide, and used after 2 weeks equilibration with daily resuspension. Cells thus prepared could be kept for several months at 4°C w i t h o u t significant loss of activity. Storage for longer periods at --20°C was possible after quick-freezing at --70°C. Sera were titrated ~sing microtitre equipment by double dilution~ e f 0.025 ml of serum in V-plates and adding 0.025 ml of 1% antigen-coated cells. End-points were read after 16 h at room temperature scoring the last positive reaction. Sera were pre-diluted 50 times and agglutination at 1/100 dilution and higher was considered positive. All dilutions were made in 1% inactivated normal rabbit serum in 0.1 M PBS at pH 7.2.
Double immunodiffusion (DID) DID was performed on 8 X 12-cm glass plates coated with a 2-mm thick layer of 1% agar gel prepared from Oxoid agar no. 1 in barbital buffer 0.04 M, pH 8.6.Alternatively, agar was made up in 10% (w/v) NaC1 in the same barbital buffer. Six wells, 5 mm in diameter, were punched at 3 mm intervals around a central well. Serial double dilutions of sera were applied to the outer wells using a 0.040-ml calibrated Hamilton syringe. The central well was filled with 0.040 ml antigen containing 4.5 protein/ml. Serum dilutions were made using a 0.050-ml microtitre system. Diffusion was performed for 48 h at room temperature and thereafter plates were washed for 48 h in 0.85% NaC1 solution. The washing fluid was replaced twice during this procedure. The agar layer was dried at 60°C under filter paper and stained for 15 min in 0.5% Coomassie brilliant blue dissolved in ethanol/water/acetic acid, 9 : 9 : 2. De-staining was carried out in the solvent until a clear background was obtained. The last dilution giving a visible precipitation line was considered as the titre of the serum.
Coun ter-immunoelectrophoresis ( CIE ) CIE was performed on glass plates as in DID with 1% agar in barbital buffer.
242 Double rows of holes, 4 m m in diameter were punched at 5-ram intervals. At the cathodic side, holes were filled with 0.025 ml diluted antigen (0.45 mg protein/ml), while in the opposite holes serum was applied. Electrophoresis was p e r f o r m e d in LKB e q u i p m e n t for 1.5 h at 7 V/cm with the cooling water kept at 20°C. Thereafter the plates were washed for 48 h in 10% NaC1 solution. Washing fluid was renewed twice during this period. The NaC1 was allowed to diffuse out of the agar into a bath containing 1% ZnSO4. This solution was used to keep weak antigen--antibody complexes precipitated. Drying and staining were p e r f o r m e d as in DID. To evaluate the sera versus the antigen, observations were scored as follows. (A) Position. The region between a n t i b o d y and antigen well was divided roughly into three equal areas: one near the serum well, a middle area, and a:~ area nearest the antigen well. Depending on the position of the particular precipitation line nearest t o the antigen well, reactions scored 1, 2 or 3 points. (B) Intensity. According to the intensity of the strongest precipitation line, reactions were scored 1 p o i n t (faint), 2 points (medium), or 3 points (intense). (C) Number of lines. Reactions were scored 1 poi nt (1 line), 2 points (2 lines), or 3 point,c, (3 or more lines). The sum of these three values was considered as the CIE value of the serum. RESULTS
Field infections in sheep In Table I the numbers and percentages of serologically positive animals are presented in relation to the fluke burdens recovered at necropsy. The n u m b e r of flukes f o u n d in the liver varied between 0 and 581. From the data presented, it is clear that IHA and CIE are more sensitive than DID. The addition of 10% NaCI to the agar lowered the n u m b e r of positive reactions although it enhanced the visibility of the lines. Even with low fluke burdens (11--40 parasites) the f o r m e r m e t hods were 100% positive. On the o th er hand, sera f r om a n u m b e r of sheep with a zero fluke c o u n t at necropsy (41 animals) were proved to be positive as well. This might be the result of non-specific reactions of u n k n o w n origin. However, anot her explanation can be p u t forward. The animals grazed in potential fluke habitats and the possibility c a n n o t be excluded t hat during their grazing period t h e y picked up some metacercariae, which were n o t de t e c t ed as flukes at necropsy. Within this premise it might be assumed t ha t the p r o p o r t i o n of positive serological determinations in these " f l u k e - f r e e " animals should be higher within grazing groups with a relatively high total fluke c o u n t as com pared with groups at a lower general level of infection. The differentiation of the serologically positive animals with " z e r o - f l u k e " counts with regard to the fluke burden in their pasturing groups as a whole (i.e. c o n c o m i t a n t animals) is shown in Table II. The hypothesis m e n t i o n e d above is confirmed by the data, i.e. chances that light fluke infections were missed at necropsy and as a result sheep were
TABLE I Serological r e a c t i o n s in r e l a t i o n t o fluke b u r d e n s in field i n f e c t i o n s in s h e e p N u m b e r o f flukes recovered
0 1--3 4--10 11--40 41--100 >100 total
Number of animals
N u m b e r o f a n i m a l s positive
P e r c e n t a g e positive a n i m a l s
IHA
CIE
DID
IHA
CIE
DID
41 32 24 16 5 4
11 21 22 16 5 4
12 24 24 16 5 4
2 14 14 7 2 3
1 8 8 5 1 3
27 66 92 100 100 100
30 75 100 100 100 100
5 44 58 44 40 75
122
79
85
42
26
D I D - - 1 0 % NaCl
DI
244
TABLE
II
Positive serological reactions in animals with zero fluke counts from flocks of four sheep infected at different levels
Total number of flukes recovered from concomitant animals
Number of "fluke-free" animals
0--1 2--6 >6
Animals serologically positive IHA
CIE
DID
DID--10t
20
2
2
0
0
13
6
4
1
2
8
3
6
0
0
e r r o n e o u s l y classified as " f l u k e - f r e e " are p r o p o r t i o n a l l y d i s t r i b u t e d w i t h t h e level o f i n f e c t i o n in t h e group. This c o u l d be t h e r e a s o n f o r the o b s e r v e d antib o d y titres. In T a b l e III, t h e r e l a t i o n s h i p b e t w e e n f l u k e b u r d e n a n d t h e level o f t h e CIE value a n d r e c i p r o c a l titre in I H A is s h o w n . In general t h e r e l a t i o n s h i p is clear, higher f l u k e b u r d e n s are a s s o c i a t e d w i t h higher titres a n d values. TABLE III Antibody titre and fluke burden in field infections in sheep Fluke burden
Number of animals with IHA reciprocal titre
CIE value
0 1--10 >10
Total
0
3--5
>5
<100
100
>100
29 8 0
8 21 5
4 27 20
30 13 0
8 18 10
3 25 15
41 56 25
37
34
51
43
36
43
122
Infection trials in sheep In Fig.1 m e a n egg c o u n t s , I H A titres a n d C I E values f o r t h e f o u r s h e e p are graphically r e p r o d u c e d . Fig.2 :illustrates t h e c o u r s e o f t h e s e p a r a m e t e r s f o r an individual sheep. F r o m t h e graphs it is clear t h a t t h e specific serological r e s p o n s e s c a n be m e a s u r e d as early as 3 w e e k s p o s t i n f e c t i o n (p.i.). A f t e r an early rise, a n d a decline at 5--6 w e e k s p.i., a n t i b o d y titres rise c o n t i n u o u s l y to r e a c h a m o r e or less c o n s t a n t level d u r i n g t h e p a t e n t p e r i o d . As the r e c o v e r y rates o f flukes in these s h e e p w e r e l o w (8, 9, 8 a n d 29 flukes) a n d faecal egg outp u t m e a s u r e d b y t h e s t a n d a r d m e t h o d s was negligible, serological t e c h n i q u e s p r o v e d valuable.
245
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16
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and CIE value in a representative sheep
Infections in cattle In Figs 3 and 4 information about the four calves is graphically depicted. The pattern in cattle is similar to that in sheep. Fluke burdens at necropsy were 19, 21, 37 and 129. DISCUSSION
From the results above it is obvious that serological analysis can provide
246
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Fig.3. Mean egg output ( - * - * - ), IHA titre ( - , ly infected calves.
•
160
180
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Fig.4. Egg output
/
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40 7
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* - ), I H A t i t r e ( - . - - o - - )
160 8
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160-'
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and CIE value in a representative
calf.
v a l u a b l e i n f o r m a t i o n a b o u t f l u k e i n f e c t i o n s in c a t t l e and s h e e p . P o s i t i v e results c a n b e m e a s u r e d earlier t h a n in f a e c a l a n a l y s i s , e v e n in sera f r o m a n i m a l s w i t h v e r y l o w f a e c a l egg c o u n t s (i.e. o n e e g g / 3 g ) . CIE a n d I H A are s u p e r i o r t o D I D , b u t in CIE d i s c r i m i n a t i o n b e t w e e n o n e or m o r e lines c a n give e x t r a i n f o r m a t i o n , as p o t e n t i a l c r o s s r e a c t i o n s are n o t l i k e l y t o p r o d u c e m o r e t h a n o n e line. In g e n e r a l , t r u e !iver f l u k e i n f e c t i o n s are c h a r a c t e r i z e d b y t w o t o
247 five lines {Nansen, 1970). Nevertheless IHA has the advantage of being easily applied to a larger number of samples by means of automation. Occasionally, however, weak positive reactions were encountered in sheep raised parasite free. Practical consequences of this p h e n o m e n o n are under investigation. For instance, greater reliability should be achieved by the use of more purified antigens. Quantitative determination by servm dilutions in CIE is too time-consuming for routine application. Moreover, the use of the suggested CIE value gives only limited information. Where serum titres are requested IHA is preferable. In agar gel precipitation techniques CIE is more sensitive than DID because diffusion of antigen and antibody is in a single direction in contrast to DID where diffusion is circular. Depending on tbe distance between the wells in DID, the increase in sensitivity is between two and four times. According to Soulsby (1967), sera from cattle and sheep give better precipitation lines at high NaC1 concentrations (10--14%). Though we could not entirely confirm this for sheep sera, it is apparently true for cattle sera. Precipitates formed under these circumstances were more stable, but when the plates are washed in physiological saline to remove non-precipitated protein, weak precipitation lines were i-edissolved. In DID, washing of plates in 10% NaC1 solution was not carried out because of the aspecific precipitation of serum proteins. In CIE where high ionic strength can only be introduced after electrophoresis, this problem does not exist because these proteins migrate to the anode and cannot interfere with antigen-antibody precipitation lines. For this reason CIE plates can be washed in 10 % NaC1. In this system false positive reactions due to C-reactive protein (Biguet et al., 1965) are eliminated as Coomassie brilliant blue does n o t stain such precipitates. Cross-reactions with other parasites were not found in serological analysis of sera from cattle artificially infected with Ostertagia ostertagi, Cooperia oncophora and Dictyo-
caulus viviparus. Experimental infections gave lower titres in cattle than in sheep. This is a well-known experience in liver fluke serology (Horchner, 1973). Of special interest was the drop in antibody titre in the calves between 20 and 40 days p.i. (Figs 3 and 4). In sheep, a similar but less pronounced phenomenon was also observed (Figs 1 and 2). This may be related to the migratory behaviour of the parasite. Low antibody titres are measured in the period which coincides roughly with the time of transition of liver flukes from the parenchyma to the bile ducts (Dargie et al., 1974). Another explanation may be that titres measured in serum are only the difference between production of antibody m~d loss of antibody by reaction with antigen. At the time of depression of antibody titre, most of the antibody would be used by the antigen which is present in the host. ACKNOWLEDGEMENTS We wish to t h a n k Misses Lenie Witter, Gerda Bijl and Mr C.P.H. Gaasenbeek for skilful technical assistance and suggestions.
248
REFERENCES Biguet, J., Capron, A., Tran Van Ky, P. and Rose, F., 1965. Pr6sence de substances de type C dans les antig~nes vermineux et de proteine anti-C au cours des helminthiases humaines ou exp~rimentales. Rev. Immunol., 29: 233--240. Boray, J.C., 1963. Standardization of techniques for pathological and anthelmintic studies with Fasciola spp. In: E.J.L. Soulsby (Editor), The Evaluation of Anthelmintics. Proceedings 1st International Conference W.A.A.V.P., Hanover. Merck, Rahway, N.J., pp. 34--45. Cho, H.J. and Ingram, D.G., 1972. Antigen and antibody in Aleutian disease in mink. I. Precipitation reactions by agar-gel electrophoresis. J. Immunol., 108: 555--557. Dargie, J.D., Armour, J., Rushton, B. and Murray, M., 1974. Immune mechanisms and hepatic fibrosis in fascioliasis. In: E.J.L. Soulsby (Editor), Parasitic Zoonoses. Academic Press, New York, N.Y., pp. 249--271. Dijkstra, J., van Vliet, G. and Over, H.J., 1973. De fasciolicide werking van "Ranide" op Fasciola .hepatica L. in experimentele infecties bij het schaap. Tijdschr. Diergeneeskd., 98: 702--712. Dorsman, W., 1956. A new technique for counting eggs of Fasciola hepatica in cattle faeces. J. Helminthol., 30: 165--172. Doyle, J.J., 1973. The precipitation response induced in calves by a single experimental infection with Fasciola hepatica. Res. Vet. Sci., 15: 119--120. Eidinger, D., 1964. A modified tanned cell haemagglutination procedure for the study of organ antigen--antibody systems. Nature (London), 201: 1046. Geyer, E., 1967. Immunology of fascioliasis. Ifi: 2rid International Liverfluke Colloquium, Wageningen, October 2nd--6th. Merck, Rahway, N.Y., pp. 46--60. Gocke, D.J. and Howe, C., 1970. Rapid detection of Australia antigen by counter-immunoelectrophoresis. J. Immunol., 104: 1031--1032. Gundlach, J.L., 1971. Studies on the serological activity of Fasciola hepatica L. antigens. Acta Parasitol. Pol., 19: 9--47. Henriksen, S.V.A.A., 1974. Fascioliasis, diagnostics. Nord. Veterinarmed., 26 (suppl. 1): 21--22. Herbert, W.J., 1967. Passive haemagglutination. In: D.M. Weir (Editor), Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford, Edinburgh, pp. 720--735. Horchner, H.W., 1973. Fasciolose Symposium 1973, Tecklenburg, October, 3rd--4th, pp. 14--18. Kabat, E.A., 1961. Estimation of protein with the biuret and ninhydrin reactions. In: E.A. Kabat and M.M. Mayer (Editors). Experimental Immunochemistry. Charles C. Thomas, Springfield, Ill., pp. 559--563. Kagan, I.K., 1974. Advances in the immunodiagnosis of parasitic infections. Z. Parasitenkd., 5: 163--195. Kent, N.H., 1960. Isolation of specific antigens from Ascaris lumbricoides. Exp. Parasitol., 10: 313--323. Koch, H.W., 1969. Untersuchungen uber die Brauchbarkeit von Haemagglutination, Flocculation, Mikro-Agar Pr~tzipitation und der Immunofluoreszens zum frtihzeitigen Nachweis der Fasciolose des Rindes. Veterinarmedizinische Dissertation, Berlin, Journal no. 671, 28 pp. Nansen, P., 1970. Metabolism of bovine immunoglobulin-G. Thesis, Copenhagen, 201 pp. Soulsby, E.J.L., 1967. Antibodies to helminths. In: D.M. Weir (Editor), Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford, Edinburgh, pp. 938--966. Trawinsky, A., 1937. iJber die Nachweis der Leberfiiule mittels der Pr~zipitin-methode. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg., Abt. 1: Orig., 139: 90--95.
SEROLOGICAL DIAGNOSIS OF FASCIOLIASIS
L.J. VAN TIGGELE and H.J. OVER Central Veterinary Institute, Lelystad (The Netherlands) (Received July 14th, 1975)
ABSTRACT Van Tiggele, L.J. and Over, H.J., 1976. Serological diagnosis of fascioliasis. Vet. Parasitol., 1: 239--248. Antibodies against Fasciola hepatica were detected in sera from experimentally infected sheep and calves. Titres in sera from field-infected sheep were related to fluke burdens. Indirect haemagglutination, counter-immunoelectrophoresis and double immunodiffusion were compared. Indirect haemagglutination and counter-immunoelectrophoresis were superior to double immunodiffusion and positive reactions in these techniques preceded the presence of parasite eggs in the faeces by at least 50 days. The relative advantages of the immunological techniques are discussed.
INTRODUCTION During t h e p a s t d e c a d e s c a t t e r e d i n f o r m a t i o n has b e c o m e available o n m e t h o d s o t h e r t h a n c o p r o l o g i c a l analysis f o r t h e diagnosis o f i n f e c t i o n w i t h Fasciola hepatica ( G e y e r , 1 9 6 7 ; G u n d l a c h , 1971). During t h e p r e p a t e n t period, a n d at l o w levels o f a d u l t f l u k e b u r d e n in p a r t i c u l a r , t h e n e e d f o r such t e c h n i q u e s is clear {H~rchner, 1 9 7 3 ; H e n r i k s e n , 1974). In e x p e r i m e n t a l i n f e c t i o n s as well as in field cases d o u b l e i m m u n o d i f f u s i o n {DID) p r o v e d t o be reliable, a l t h o u g h s o m e w h a t insensitive ( T r a w i n s k y , 1 9 3 7 ; K o c h , 1 9 6 9 ; Doyle, 1973). T o o b t a i n higher sensitivity we a d o p t e d c o u n t e r - i m m u n o e l e c t r o p h o r e s i s (CIE) a n d i n d i r e c t h a e m a g g l u t i n a t i o n ( I H A ) . C I E is an i m p r o v e m e n t on D I D a n d has also b e e n a p p l i e d t o c e r t a i n viral diseases s u c h as s e r u m h e p a t i t i s in m a n a n d A l e u t i a n disease in m i n k ( G o c k e and Howe, 1970; Cho and Ingram, 1972). I H A is a useful m e t h o d f o r large scale serological analysis in m i c r o t i t r e s y s t e m s . I t has b e e n a p p l i e d in a wide r a n g e o f parasitic i n f e c t i o n s in m a n (Kagan, 1 9 7 4 ) . Since serological m e t h o d s c a n b e s i m p l y u s e d t o t e s t a large n u m b e r o f sera, a t t e m p t s w e r e m a d e t o i m p r o v e a n d e v a l u a t e t h e s e useful t e c h n i q u e s .
240
MATERIAL AND METHODS
Hosts Blood samples were collected at necropsy from 122 Texel sheep, 7--14 months of age. They had grazed previously in groups of four for 2--3 months on fields with different infection levels and were kept for 3 months on fluke-free fields after this period. Thus at necropsy the infections were 3--6 months old. Flukes were collected from these sheep using previously described techniques (Dijkstra et al., 1973). To analyse the development of antibody titres during the course of infection, four 1-year-old sheep raised parasite free were infected with 500 metacercariae each. Infection was monitored by regular faecal examination and standard haematological procedures. During the period following infection, serum samples were analysed weekly for 8 weeks, then every 3 weeks for 18 weeks. After 26 weeks the sheep were killed and flukes were recovered and counted. In an experiment on calves, four 8-month-old animals were infected with 2 000 metacercariae each. The animals were kept in normal farm conditions on fluke-free fields. During the course of the experiment, observations were made every 10 or 11 days with regard to egg production, serology and haematology. After 15 weeks serological examination was performed every 21 days. The animals were necropsied after 26 weeks and flukes were collected. All sera were stored at --20°C until examination.
Infective material In the artificial infection experiment, a fluke strain obtained by cloning was used. Cercariae were obtained from Lymnaea truncatula at 21°C. They were collected according to Boray's (1963) method, but instead of encysting on glass the metacercariae were collected on cellophane paper. Metacercariae were stored at 4°C {relative h u m i d i t y 100%) and were 3--8 weeks of age at the time of infection. Animals were infected orally by administration of gelatine capsules containing the metacercariae.
Faecal examination Faeces samples were examined according to the m e t h o d described by Dorsman {1956).
Antigens Flukes were obtained from sheep livers at a slaughterhouse and a standard lipid-free antigen was prepared according to Kent (1960).
241 Protein c o n t e n t was estimated using the biuret m e t h o d as described by Kabat (1961). Antigen containing 4.5 mg protein/ml was stored a t - - 2 0 ° C t h r o u g h o u t the experiment.
Indirect haemagglutination (IHA ) Sheep erythrocytes were fixed as described by Herbert (1967) using glutaraldehyde instead of formaldehyde. A 1% suspension of these stabilized cells was sensitized with tannic acid 1 : 40 000. To prevent clumping, tanned cells were pre-coated by mixing equal volumes of 2% tanned cells in 0.1 M phosphate buffered saline (PBS) pH 7.2 with 0.5% bovine serum albumin in PBS (Eidinger, 1964). Antigens were attached at pH 6.4 using 0.2 ml of standard antigen (4.5 mg/ml) per 10 ml of 1% cells (30 min, 37°C). Antigen-coated cells were stored at 4°C in 1% inactivated normal rabbit serum in PBS with 0.02% sodium azide, and used after 2 weeks equilibration with daily resuspension. Cells thus prepared could be kept for several months at 4°C w i t h o u t significant loss of activity. Storage for longer periods at --20°C was possible after quick-freezing at --70°C. Sera were titrated ~sing microtitre equipment by double dilution~ e f 0.025 ml of serum in V-plates and adding 0.025 ml of 1% antigen-coated cells. End-points were read after 16 h at room temperature scoring the last positive reaction. Sera were pre-diluted 50 times and agglutination at 1/100 dilution and higher was considered positive. All dilutions were made in 1% inactivated normal rabbit serum in 0.1 M PBS at pH 7.2.
Double immunodiffusion (DID) DID was performed on 8 X 12-cm glass plates coated with a 2-mm thick layer of 1% agar gel prepared from Oxoid agar no. 1 in barbital buffer 0.04 M, pH 8.6.Alternatively, agar was made up in 10% (w/v) NaC1 in the same barbital buffer. Six wells, 5 mm in diameter, were punched at 3 mm intervals around a central well. Serial double dilutions of sera were applied to the outer wells using a 0.040-ml calibrated Hamilton syringe. The central well was filled with 0.040 ml antigen containing 4.5 protein/ml. Serum dilutions were made using a 0.050-ml microtitre system. Diffusion was performed for 48 h at room temperature and thereafter plates were washed for 48 h in 0.85% NaC1 solution. The washing fluid was replaced twice during this procedure. The agar layer was dried at 60°C under filter paper and stained for 15 min in 0.5% Coomassie brilliant blue dissolved in ethanol/water/acetic acid, 9 : 9 : 2. De-staining was carried out in the solvent until a clear background was obtained. The last dilution giving a visible precipitation line was considered as the titre of the serum.
Coun ter-immunoelectrophoresis ( CIE ) CIE was performed on glass plates as in DID with 1% agar in barbital buffer.
242 Double rows of holes, 4 m m in diameter were punched at 5-ram intervals. At the cathodic side, holes were filled with 0.025 ml diluted antigen (0.45 mg protein/ml), while in the opposite holes serum was applied. Electrophoresis was p e r f o r m e d in LKB e q u i p m e n t for 1.5 h at 7 V/cm with the cooling water kept at 20°C. Thereafter the plates were washed for 48 h in 10% NaC1 solution. Washing fluid was renewed twice during this period. The NaC1 was allowed to diffuse out of the agar into a bath containing 1% ZnSO4. This solution was used to keep weak antigen--antibody complexes precipitated. Drying and staining were p e r f o r m e d as in DID. To evaluate the sera versus the antigen, observations were scored as follows. (A) Position. The region between a n t i b o d y and antigen well was divided roughly into three equal areas: one near the serum well, a middle area, and a:~ area nearest the antigen well. Depending on the position of the particular precipitation line nearest t o the antigen well, reactions scored 1, 2 or 3 points. (B) Intensity. According to the intensity of the strongest precipitation line, reactions were scored 1 p o i n t (faint), 2 points (medium), or 3 points (intense). (C) Number of lines. Reactions were scored 1 poi nt (1 line), 2 points (2 lines), or 3 point,c, (3 or more lines). The sum of these three values was considered as the CIE value of the serum. RESULTS
Field infections in sheep In Table I the numbers and percentages of serologically positive animals are presented in relation to the fluke burdens recovered at necropsy. The n u m b e r of flukes f o u n d in the liver varied between 0 and 581. From the data presented, it is clear that IHA and CIE are more sensitive than DID. The addition of 10% NaCI to the agar lowered the n u m b e r of positive reactions although it enhanced the visibility of the lines. Even with low fluke burdens (11--40 parasites) the f o r m e r m e t hods were 100% positive. On the o th er hand, sera f r om a n u m b e r of sheep with a zero fluke c o u n t at necropsy (41 animals) were proved to be positive as well. This might be the result of non-specific reactions of u n k n o w n origin. However, anot her explanation can be p u t forward. The animals grazed in potential fluke habitats and the possibility c a n n o t be excluded t hat during their grazing period t h e y picked up some metacercariae, which were n o t de t e c t ed as flukes at necropsy. Within this premise it might be assumed t ha t the p r o p o r t i o n of positive serological determinations in these " f l u k e - f r e e " animals should be higher within grazing groups with a relatively high total fluke c o u n t as com pared with groups at a lower general level of infection. The differentiation of the serologically positive animals with " z e r o - f l u k e " counts with regard to the fluke burden in their pasturing groups as a whole (i.e. c o n c o m i t a n t animals) is shown in Table II. The hypothesis m e n t i o n e d above is confirmed by the data, i.e. chances that light fluke infections were missed at necropsy and as a result sheep were
TABLE I Serological r e a c t i o n s in r e l a t i o n t o fluke b u r d e n s in field i n f e c t i o n s in s h e e p N u m b e r o f flukes recovered
0 1--3 4--10 11--40 41--100 >100 total
Number of animals
N u m b e r o f a n i m a l s positive
P e r c e n t a g e positive a n i m a l s
IHA
CIE
DID
IHA
CIE
DID
41 32 24 16 5 4
11 21 22 16 5 4
12 24 24 16 5 4
2 14 14 7 2 3
1 8 8 5 1 3
27 66 92 100 100 100
30 75 100 100 100 100
5 44 58 44 40 75
122
79
85
42
26
D I D - - 1 0 % NaCl
DI
244
TABLE
II
Positive serological reactions in animals with zero fluke counts from flocks of four sheep infected at different levels
Total number of flukes recovered from concomitant animals
Number of "fluke-free" animals
0--1 2--6 >6
Animals serologically positive IHA
CIE
DID
DID--10t
20
2
2
0
0
13
6
4
1
2
8
3
6
0
0
e r r o n e o u s l y classified as " f l u k e - f r e e " are p r o p o r t i o n a l l y d i s t r i b u t e d w i t h t h e level o f i n f e c t i o n in t h e group. This c o u l d be t h e r e a s o n f o r the o b s e r v e d antib o d y titres. In T a b l e III, t h e r e l a t i o n s h i p b e t w e e n f l u k e b u r d e n a n d t h e level o f t h e CIE value a n d r e c i p r o c a l titre in I H A is s h o w n . In general t h e r e l a t i o n s h i p is clear, higher f l u k e b u r d e n s are a s s o c i a t e d w i t h higher titres a n d values. TABLE III Antibody titre and fluke burden in field infections in sheep Fluke burden
Number of animals with IHA reciprocal titre
CIE value
0 1--10 >10
Total
0
3--5
>5
<100
100
>100
29 8 0
8 21 5
4 27 20
30 13 0
8 18 10
3 25 15
41 56 25
37
34
51
43
36
43
122
Infection trials in sheep In Fig.1 m e a n egg c o u n t s , I H A titres a n d C I E values f o r t h e f o u r s h e e p are graphically r e p r o d u c e d . Fig.2 :illustrates t h e c o u r s e o f t h e s e p a r a m e t e r s f o r an individual sheep. F r o m t h e graphs it is clear t h a t t h e specific serological r e s p o n s e s c a n be m e a s u r e d as early as 3 w e e k s p o s t i n f e c t i o n (p.i.). A f t e r an early rise, a n d a decline at 5--6 w e e k s p.i., a n t i b o d y titres rise c o n t i n u o u s l y to r e a c h a m o r e or less c o n s t a n t level d u r i n g t h e p a t e n t p e r i o d . As the r e c o v e r y rates o f flukes in these s h e e p w e r e l o w (8, 9, 8 a n d 29 flukes) a n d faecal egg outp u t m e a s u r e d b y t h e s t a n d a r d m e t h o d s was negligible, serological t e c h n i q u e s p r o v e d valuable.
245
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16
18 9
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and CIE value in a representative sheep
Infections in cattle In Figs 3 and 4 information about the four calves is graphically depicted. The pattern in cattle is similar to that in sheep. Fluke burdens at necropsy were 19, 21, 37 and 129. DISCUSSION
From the results above it is obvious that serological analysis can provide
246
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Fig.3. Mean egg output ( - * - * - ), IHA titre ( - , ly infected calves.
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v a l u a b l e i n f o r m a t i o n a b o u t f l u k e i n f e c t i o n s in c a t t l e and s h e e p . P o s i t i v e results c a n b e m e a s u r e d earlier t h a n in f a e c a l a n a l y s i s , e v e n in sera f r o m a n i m a l s w i t h v e r y l o w f a e c a l egg c o u n t s (i.e. o n e e g g / 3 g ) . CIE a n d I H A are s u p e r i o r t o D I D , b u t in CIE d i s c r i m i n a t i o n b e t w e e n o n e or m o r e lines c a n give e x t r a i n f o r m a t i o n , as p o t e n t i a l c r o s s r e a c t i o n s are n o t l i k e l y t o p r o d u c e m o r e t h a n o n e line. In g e n e r a l , t r u e !iver f l u k e i n f e c t i o n s are c h a r a c t e r i z e d b y t w o t o
247 five lines {Nansen, 1970). Nevertheless IHA has the advantage of being easily applied to a larger number of samples by means of automation. Occasionally, however, weak positive reactions were encountered in sheep raised parasite free. Practical consequences of this p h e n o m e n o n are under investigation. For instance, greater reliability should be achieved by the use of more purified antigens. Quantitative determination by servm dilutions in CIE is too time-consuming for routine application. Moreover, the use of the suggested CIE value gives only limited information. Where serum titres are requested IHA is preferable. In agar gel precipitation techniques CIE is more sensitive than DID because diffusion of antigen and antibody is in a single direction in contrast to DID where diffusion is circular. Depending on tbe distance between the wells in DID, the increase in sensitivity is between two and four times. According to Soulsby (1967), sera from cattle and sheep give better precipitation lines at high NaC1 concentrations (10--14%). Though we could not entirely confirm this for sheep sera, it is apparently true for cattle sera. Precipitates formed under these circumstances were more stable, but when the plates are washed in physiological saline to remove non-precipitated protein, weak precipitation lines were i-edissolved. In DID, washing of plates in 10% NaC1 solution was not carried out because of the aspecific precipitation of serum proteins. In CIE where high ionic strength can only be introduced after electrophoresis, this problem does not exist because these proteins migrate to the anode and cannot interfere with antigen-antibody precipitation lines. For this reason CIE plates can be washed in 10 % NaC1. In this system false positive reactions due to C-reactive protein (Biguet et al., 1965) are eliminated as Coomassie brilliant blue does n o t stain such precipitates. Cross-reactions with other parasites were not found in serological analysis of sera from cattle artificially infected with Ostertagia ostertagi, Cooperia oncophora and Dictyo-
caulus viviparus. Experimental infections gave lower titres in cattle than in sheep. This is a well-known experience in liver fluke serology (Horchner, 1973). Of special interest was the drop in antibody titre in the calves between 20 and 40 days p.i. (Figs 3 and 4). In sheep, a similar but less pronounced phenomenon was also observed (Figs 1 and 2). This may be related to the migratory behaviour of the parasite. Low antibody titres are measured in the period which coincides roughly with the time of transition of liver flukes from the parenchyma to the bile ducts (Dargie et al., 1974). Another explanation may be that titres measured in serum are only the difference between production of antibody m~d loss of antibody by reaction with antigen. At the time of depression of antibody titre, most of the antibody would be used by the antigen which is present in the host. ACKNOWLEDGEMENTS We wish to t h a n k Misses Lenie Witter, Gerda Bijl and Mr C.P.H. Gaasenbeek for skilful technical assistance and suggestions.
248
REFERENCES Biguet, J., Capron, A., Tran Van Ky, P. and Rose, F., 1965. Pr6sence de substances de type C dans les antig~nes vermineux et de proteine anti-C au cours des helminthiases humaines ou exp~rimentales. Rev. Immunol., 29: 233--240. Boray, J.C., 1963. Standardization of techniques for pathological and anthelmintic studies with Fasciola spp. In: E.J.L. Soulsby (Editor), The Evaluation of Anthelmintics. Proceedings 1st International Conference W.A.A.V.P., Hanover. Merck, Rahway, N.J., pp. 34--45. Cho, H.J. and Ingram, D.G., 1972. Antigen and antibody in Aleutian disease in mink. I. Precipitation reactions by agar-gel electrophoresis. J. Immunol., 108: 555--557. Dargie, J.D., Armour, J., Rushton, B. and Murray, M., 1974. Immune mechanisms and hepatic fibrosis in fascioliasis. In: E.J.L. Soulsby (Editor), Parasitic Zoonoses. Academic Press, New York, N.Y., pp. 249--271. Dijkstra, J., van Vliet, G. and Over, H.J., 1973. De fasciolicide werking van "Ranide" op Fasciola .hepatica L. in experimentele infecties bij het schaap. Tijdschr. Diergeneeskd., 98: 702--712. Dorsman, W., 1956. A new technique for counting eggs of Fasciola hepatica in cattle faeces. J. Helminthol., 30: 165--172. Doyle, J.J., 1973. The precipitation response induced in calves by a single experimental infection with Fasciola hepatica. Res. Vet. Sci., 15: 119--120. Eidinger, D., 1964. A modified tanned cell haemagglutination procedure for the study of organ antigen--antibody systems. Nature (London), 201: 1046. Geyer, E., 1967. Immunology of fascioliasis. Ifi: 2rid International Liverfluke Colloquium, Wageningen, October 2nd--6th. Merck, Rahway, N.Y., pp. 46--60. Gocke, D.J. and Howe, C., 1970. Rapid detection of Australia antigen by counter-immunoelectrophoresis. J. Immunol., 104: 1031--1032. Gundlach, J.L., 1971. Studies on the serological activity of Fasciola hepatica L. antigens. Acta Parasitol. Pol., 19: 9--47. Henriksen, S.V.A.A., 1974. Fascioliasis, diagnostics. Nord. Veterinarmed., 26 (suppl. 1): 21--22. Herbert, W.J., 1967. Passive haemagglutination. In: D.M. Weir (Editor), Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford, Edinburgh, pp. 720--735. Horchner, H.W., 1973. Fasciolose Symposium 1973, Tecklenburg, October, 3rd--4th, pp. 14--18. Kabat, E.A., 1961. Estimation of protein with the biuret and ninhydrin reactions. In: E.A. Kabat and M.M. Mayer (Editors). Experimental Immunochemistry. Charles C. Thomas, Springfield, Ill., pp. 559--563. Kagan, I.K., 1974. Advances in the immunodiagnosis of parasitic infections. Z. Parasitenkd., 5: 163--195. Kent, N.H., 1960. Isolation of specific antigens from Ascaris lumbricoides. Exp. Parasitol., 10: 313--323. Koch, H.W., 1969. Untersuchungen uber die Brauchbarkeit von Haemagglutination, Flocculation, Mikro-Agar Pr~tzipitation und der Immunofluoreszens zum frtihzeitigen Nachweis der Fasciolose des Rindes. Veterinarmedizinische Dissertation, Berlin, Journal no. 671, 28 pp. Nansen, P., 1970. Metabolism of bovine immunoglobulin-G. Thesis, Copenhagen, 201 pp. Soulsby, E.J.L., 1967. Antibodies to helminths. In: D.M. Weir (Editor), Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford, Edinburgh, pp. 938--966. Trawinsky, A., 1937. iJber die Nachweis der Leberfiiule mittels der Pr~zipitin-methode. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg., Abt. 1: Orig., 139: 90--95.